The retinoblastoma tumor suppressor gene is a prototype for tumor suppressor genes. Its protein, pRB, is an integral protein that is disrupted in a vast majority of human tumors. Understanding the underlying mechanisms of pRB tumor suppression is, therefore, of critical importance. What is the molecular mechanism of pRB action? In various experimental settings, pRB can be shown to mediate cell cycle arrest, promote cellular differentiation, suppress apoptosis, repress transcription of some transcription factors (E2F) and activate transcription with others (myoD and C/EBP). These activities have been studied primarily in artificial assay systems and currently it is unclear which of these pRB properties are most important in-vivo. It is known that a large domain of pRB, the pocket, is necessary and sufficient for most of pRB's activities. Studies of chimeric mice pith RB -/- cells demonstrate that loss of pRB does not remove most aspects of cell cycle control. In such mice, RB -/- cells contribute to most tissues and have normal histology. We hypothesize, that pRB is not generally essential for cell cycle control but that it must have very specific roles relevant to tumorigenesis. Preliminary data from the Dyson laboratory suggests that maintenance of the genomic integrity of cells may be dependent upon pRB's ability to induce a cell cycle arrest in response to DNA damage. Recent findings also suggest that pRB's ability to promote differentiation correlates with tumorigenicity. This proposal will investigate the in-vivo significance of pRB pathways leading to cell cycle arrest in response to DNA damage and to cellular differentiation in pRB tumor suppression. Specific Aim number 1: To identify the structural features important for pRB to induce cell cycle arrest in response to DNA damage and to promote cellular differentiation. The pRB-associated proteins essential to these two pathways will be identified. Specific Aim number 2: To determine whether the ability of pRB to arrest cell cycle and promote cellular differentiation is important for pRB to act as a tumor suppressor in-vivo using the RB mutants generated in aim number 1 to disable these specific pathways. Specific Aim number 3: To determine whether the deregulation of E2F dependent transcription is correlated with the tumorigenic potential of RB -/- cells in-vivo.